The regulated expression of ICAM-l by microvascular endothelial cells (HDMEC) and keratinocytes (HK) is vital to the initiation and evolution of localized cutaneous inflammation. We have determined that ICAM-l is differentially regulated in HK and HDMEC, and that induction of ICAM-l by IFN-gamma is mediated through a novel trans-acting protein complex that activates transcription via a unique IFN-gamma response element, labeled pIgammaRE. Recent evidence indicates that induction of many genes by IFN- gamma involves members of a newly described family of proteins. These factors are critical to both signal transduction across cell membranes and activation of transcription and are thus collectively referred to as signal transducers and activators of transcription, or STAT proteins. While the initial descriptions of STAT proteins have utilized lymphoid cell lines, it is clear that IFN-gamma mediates many of its effects in cutaneous inflammation through cells, such as HK and HDMEC, that are unique to the skin. However, little is known about the expression and function of STAT-related proteins within these cells. We present preliminary data indicating that the trans-acting factors which bind to the ICAM-l pIgammaRE comprise a novel STAT-related protein complex. We hypothesize that the profile of genes activated by IFN-gamma in HK and HDMEC is dependent upon the differential expression, activation, and function of tissue-specific STAT proteins. Using the ICAM-l pIgammaRE as our focus for investigation, we propose to characterize the complement of STAT-related proteins expressed and activated in HK and HDMEC in response to IFN-gamma, and to characterize the biology and molecular mechanisms by which specific STAT-related proteins regulate the expression of IFN-gamma responsive genes, such as ICAM-1, in these cells.
Our Specific Aims are: l) To characterize the expression, activation, and functional activity of STAT-related proteins which bind and activate transcription via the ICAM-l pIgammaRE in HK and HDMEC in response to IFN-gamma stimulation; 2) To characterize the profile of expression of known STAT-related proteins and their activation by IFN-gamma in HK and HDMEC; 3) To characterize and purify components of the STAT-related protein complex expressed by HK and HDMEC that bind and transactivate gene expression via the ICAM-1 pIgammaRE; 4) To isolate and characterize cDNAs encoding novel pIgammaRE binding STAT-related proteins that are activated in HK and HDMEC by IFN- gamma stimulation; and 5) To characterize the expression, activation and function of these novel IFN-gamma-dependent STAT proteins expressed by HK and HDMEC. Delineation and characterization of these signaling and regulatory proteins, their abilities and functions in inducing specific gene transcription, and the regulation of their expression and activation within these two distinct cellular populations will provide critical insights into our understanding of the molecular events involved in cutaneous inflammatory and immunologic processes.
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